The use of solution NMR spectroscopy to analyze the dynamics, interactions and function of large proteins (>100 kDa) and supra-molecular systems is becoming increasingly feasible. Key to this advance has been the development of new and powerful ways of labeling biomolecules has been critical for stimulating many of the advances in NMR methodology.
Methyl groups have been proven to be ideal molecular probes for solution NMR spectroscopy studies of large proteins.
In initial methyl-labeling procedures, alpha-keto acids were used as precursors in the production of methyl-protonated isoleucine (Ile). A more recent strategy is based on the use of labelled 2-(S)-2-hydroxy-2-ethyl-3-oxobutanoate, a biosynthetic precursor of isoleucine in E. Coli (Ayala I. et al., Chem Comm., 2011, www.rsc.org/chemcomm, DOI: 10.1039/C1CC12932E). In this method, the precursor is chemically synthetized meaning that the precursor is obtained as a racemic mixture and that only half of the mixture obtained can be converted by the bacteria.
Valine (Val), leucine (Leu) and isoleucine (Ile) are three amino acids of great interest as their methyl groups account for more than 50% of all methyl probes available in proteins.
Protonation of leucine and valine methyl groups in perdeuterated proteins is commonly achieved using methyl protonated 2-oxo-3-methylbutanoic acid (also known as alpha-ketoisovalerate), an intermediate in the biosynthesis of these amino acids, in which both methyl groups are 1H, 13C-labeled. The use of this type of alpha-ketoisovalerate proved inefficient in high-molecular-weight proteins as it results in overcrowded [1H,13C]-correlated spectra due to the sheer number of NMR-visible methyl probes.
WO 2011/083356 describes a process for the specific isotopic labeling of Valine (Val), leucine (Leu) and isoleucine (Ile) in protein assemblies using a racemic mixture of acetolactate derivatives obtained by chemical synthesis. Resolution of said racemic mixtures into their stereochemically pure acetolactate derivatives by conventional methods proved ineffective.
Ruschak A. M. et al., J Biomol NMR, 2010, 48(3), p. 129-35 and Ayala I. et al., J. Chem Commun, 2012, 48, p. 1434-1436 describe synthetic routes for preparing ester derivatives of 2-hydroxy-2-methyl-3-oxobutanoic acid and 2-hydroxy-2-ethyl-3-oxobutanoate for the specific labeling of Isoleucine gamma-2 methyl groups. In these references, the compounds are prepared by chemical synthesis from methyl (or ethyl) acetoacetate and are thus obtained in the form of a racemic mixture. Consequently, only half of the compound obtained with the S stereochemistry—can be incorporated by the bacteria. Moreover, the derivatives of 2-hydroxy-2-methyl-3-oxobutanoic acid and 2-hydroxy-2-ethyl-3-oxobutanoate are in the form of an ester, meaning that an additional step is required to deprotect the compounds in basic medium before use. Under poorly controlled reaction conditions, this may result in a significant degradation of the compounds.
Godoy-Ruiz R. et al., J. Am. Chem. Soc., 2010, 132(51), p. 18340-50 describe simultaneous selective isotope labeling of Alanine, Leucine, Valine and Isoleucine methyl positions using alpha-ketoisovalerate (for labeling Ala, Leu and Val sites) and alpha-ketobutyrate (for labeling of Ile positions) and their use to obtain distance restraints and mobility data. The labeling method described in this reference leads to isotopic leaks in the gamma-2 position of Isoleucines resulting in artifacts in the extraction of structural constraints. This phenomenon is due to the deamination of some of the labeled 3-13C-alanine leading to the in viva synthesis of labeled pyruvate. The condensation of the thus obtained labeled pyruvate with endogenous 2-oxobutanoate in the presence of the acetolactate synthase, yields 2-hydroxy-2-ethyl-3-oxo-4-13C-butanoate.
Engel, S. et al., Biotechnology & Bioengineering, 88, p. 825-83, reports the use of acetohydroxyacid synthase I (AHAS I) from Escherichia Coli in the stereoselective synthesis of aromatic alpha-hydroxy ketones.
US 2006/0148042 relates to a biotransfounation process for the preparation of chiral aromatic alpha-hydroxy ketones using acetohydroxyacid synthase (AHAS) or tartronate semialdehyde synthase (TSAS). This document is totally silent regarding the synthesis of non aromatic synthesis of chiral acetohydroxy acids, the synthesis of chiral acetohydroxy acids specifically or entirely deuterated and/or enriched in carbon 13 (13C) and their use for isotopic labeling of amino acids.
Thus, there remains a need for labeled stereospecific alpha-hydroxyl ketoacids that are capable of efficiently and specifically label amino acids, in particular the methyl groups of amino acids selected from leucine, valine and isoleucine.
In particular, there remains a need for labeled stereospecific alpha-hydroxy ketoacids as described above, that are manufactured by a regioselective and stereospecific process in high yields and under mild conditions.
More particularly, there remains a need for labeled stereospecific alpha-hydroxy ketoacids as described above, capable of labeling the amino acid without causing isotopic leaks at the site where the amino acid is labeled, i.e. the methyl group of amino acids leucine, valine and isoleucine.
Even more particularly, there remains a need for labeled stereospecific alpha-hydroxy ketoacids as described above, that can be incorporated into the target protein by the bacteria without detectable scrambling.